In general, the present invention relates to engineered transglutaminase polypeptides, methods for their production as well as for their use for binding or for recognizing given ligands.
The adaptive immune system is a highly evolved, flexible system for the recognition and neutralization of foreign organisms and macromolecules. At the core of adaptive immunity is an engine for the creation of a vast variety of different similar structures that have been diversified by combinatorial assembly of varied building blocks with highly random linker segments. The two principle recognition complexes of the higher vertebrate adaptive immune system, antibodies and the T cell antigen receptor, are similarly assembled, and function through their cognate cell types, B cells and T cells, to effect a coordinated resistance to pathogens. Although all elements of the adaptive recognition system of higher vertebrates are based on assemblies of monomer domains of the immunoglobulin fold, in cyclostomes, convergent evolution has created an adaptive immune system that is constructed by the assembly of recognition elements derived from leucine rich repeats.
The effector proteins of the B cell arm of the adaptive immune system, particularly antibodies of the IgG subtype, have many attractive properties as candidate therapeutic agents. IgG antibodies are stable highly soluble proteins with a long in vivo half life that have weak immunogenicity within a given species. They often can be selected to have high affinities for their targets and are known to have few intrinsic safety liabilities. IgG antibodies as a class have relatively predictable behavior in vitro and in vivo, but are large, heterodimeric, disulfide-stabilized, glycosylated proteins that are difficult to make in prokaryotic cells. It has been hypothesized that antibodies may be effectively replaced for a variety of purposes by artificial antibody-like proteins, derived by the diversification of natural or unnatural scaffolds. Such antibody equivalents might be more readily manufactured and might have favorable tissue penetration and biodistribution properties compared with antibodies themselves.
In recent years recombinant antibodies of substantially human sequence have played a major role in therapeutic medicine as universal recognition moieties for a number of targets in different diseases. Human monospecific antibodies of the IgG subtype provide high specificity, bivalency, fully human composition, and long plasma half-life. The known limitations of antibodies relate largely to their biophysical properties (high molecular weight, multidomain assemblage, disulfide bonds, glycosylation), which require eukaryotic manufacturing processes that are more complex and more expensive than their prokaryotic counterparts. Fragments of antibodies, such as scFv domains, Fab domains and multivalent miniantibodies have been produced in bacteria, and offer some opportunities for the realization of low cost, highly effective therapeutic agents.
Scaffolds based on different human or non-human proteins or protein domains have emerged as an independent class of alternative therapeutic molecules. The status of alternative scaffolds and selection procedures used to identify high affinity binding proteins based on those scaffolds have been recently reviewed. Different proteins have been investigated as frameworks for bringing the diversified sequences to targets, including affibodies, lipocalins, ankyrin-repeat proteins, natural peptide binding domains, enzymes, GFP, small disulfide-bonded peptides, protease inhibitors, and others. Approximately 50 protein scaffolds have been proposed so far but only a few have been developed extensively for medical applications (Adnectins (Bristol-Myers Squibb Co), Anticalins (Pieris AG), Microbodies (Nascacell Technologies AG), Nanobodies (Ablynx), Kunitz domains (Dyax), Peptide aptamers (Aptanomics), Affibodies (Affibody AB), DARPins (Molecular Partners AG), Affilins (Scil Proteins GmbH), Tetranectins (Borean) and Avimers (Amgen)). Several are in preclinical development and a few examples are undergoing clinical trials (anti-VEGFR2 AdNectin (phase I), anti-IL6 Avimer (phase I) and engineered Kunitz-type protease inhibitor anti-kallikrein DX-88 (phase II-III)).
Although for prospective therapeutic applications to date, alternative scaffolds have largely been employed as neutralizing agents for ligand-receptor interaction, cytokine, toxin, or Fc-fusions are being investigated to confer on the binding protein a cytostatic or cytotoxic effect similar to that achieved through antibody-dependent cellular cytotoxicity (ADCC). The potential role of alternative scaffolds in diagnosis is important since large arrays of specific small reagents could be produced to many different targets. Compared to antibodies, small scaffolds should have better tissue penetration which could be advantageous for solid tumor targets.
Criteria for choosing an appropriate alternative scaffold for therapeutic purposes have been disclosed by several sources. Preferable alternative scaffolds have small size (for stability, ease of manufacturing, convenience of selection in some display methods, and tissue penetration in solid tumor applications); high thermodynamic stability and high solubility (for optimal prolonged performance in human plasma) and compatibility with therapeutic use in humans. The latter has been interpreted by some to mean that the scaffold is preferably of human origin (to avoid unwanted immunogenic effects), but scaffolds based on non-human mammalian proteins, bacterial proteins, or synthetic proteins have been proposed. Preferable scaffolds often have few disulfide bonds and free cysteines (which can lead to non-specific target binding during selection), but if the scaffold fold is stable and self-associates well in prokaryotes, as described for the type A repeats disclosed by WO 06/055689, the incorporation of cysteines may not be problematic. If the scaffold is chosen to be of human origin to minimize the adverse consequences of the generation of antibodies against the scaffold, the protein to be used as an alternative scaffold should preferably already exist in human plasma, preferably at a high concentration, and the introduction of a low titer of autoreactive antibodies to the scaffold should preferably have minimal adverse physiological consequences.
The presence of a structurally rigid core that is able to tolerate changes of surface residues without losing stability or correct folding of the protein is also desirable. Alternative scaffolds preferably exhibit protease resistance in addition to their other properties. Protease resistance can be useful for manufacturing, stability, and compatibility with biological samples or environments.